For over a decade, the budding yeast Kluyveromyces lactis (K. lactis) has been widely used for industrial-scale production of recombinant proteins in the food and dairy industries for reasons that include the following factors: (i) many strains of K. lactis grow rapidly and to extremely high cell densities in culture; (ii) K. lactis efficiently directs proteins to be secreted into the medium; and (iii) K. lactis has GRAS (Generally Regarded As Safe) FDA status which permits its use for food, agricultural and health-related applications.
A typical K. lactis heterologous protein production strategy involves directing a desired protein to be secreted from the cell into the growth medium. This methodology has a number of advantages over cellular expression methods: (i) the protein is produced significantly pure since K. lactis secretes relatively few endogenous proteins; (ii) post-translational protein modifications found only on secreted eukaryotic proteins are obtainable; and (iii) strategies to harvest protein from the medium of continuously growing cells can be devised.
A strong yeast promoter suitable for directing high levels of transcription in K. lactis is the K. lactis LAC4 promoter (PLAC4) (Dickson, et al. Cell 15:123-130 (1978); Dickson, R. C., and M. I. Riley, Biotechnology 13:19-40 (1989); Dickson, et al. Mol. Cel. Biol. 1:1048-1056 (1981)). This promoter naturally drives expression of the LAC4 gene which encodes a highly expressed lactase (β-galactosidase). Transcription of LAC4 is elevated in response to the presence of lactose or galactose in growth medium where lactase allows the cell to convert lactose to fermentable sugars. Expression of heterologous proteins from PLAC4 may achieve levels greater than 100 mg L−1 of secreted recombinant protein in yeast fermentations.
Unfortunately, in addition to its ability to function as a strong promoter in K. lactis, PLAC4 constitutively promotes gene expression in E. coli cells. This can be particularly problematic when trying to assemble DNA constructs harboring genes that encode a protein toxic to E. coli prior to their introduction into yeast cells. One approach to solving this problem has been reported by Gibbs et al. (FEMS Yeast Research 4: 573-577 (2004)) who utilized yeast introns in the shuttle vector. Unfortunately, this modification abolishes some but not all functional expression of potentially toxic recombinant proteins.